Klinik & Poliklinik für Nuklearmedizin
TUM Klinikum Rechts der Isar
ph Biosensors

"Resolving spatiotemporal pH changes non-invasively in vivo using hyperpolarized pH sensor compounds"

Many efforts have been undertaken to develop an imaging method to characterize hypoxic and acidic regions within tumors, namely a quantitative non-invasive pH imaging technique. However, so far, no technique to non-invasively image extracellular pH has been applicable routinely in the clinic. In humans, pH is mainly regulated by the carbon dioxide/bicarbonate buffer within a narrow pH range, in the blood between pH 7.35 and 7.45. Locally, deviations from the systemic pH are often caused by pathologies, such as cancer, inflammation, infection, ischemia, renal failure or pulmonary disease. Our team identified the pH-sensitive molecule [1,5-13C2]zymonic acid (ZA) which we recently reported in a publication in Nature Communications (Figure 1).

Figure 1. Chemical structure, mechanism and calibration of the pH dependency of ZA at 7 T. a, ZA bears two exchangeable protons, one in a carboxy group with an acid dissociation constant pKa1 = 2.35 and a second one in an enolic hydroxy group with pKa2 = 6.90. b, Spectra of hyperpolarized ZA and urea in buffer phantoms at different pH values. The pH sensitivity in the physiological range is due to pKa2 and results in a change of the chemical shifts of the two 13C-labeled carbon positions with respect to the pH-insensitive 13C-urea peak as a function of pH. Additional peaks of a decay product of ZA, parapyruvate hydrate (PPH), can be identified. c, The fit of the data from (b) results in a calibration curve with a direct dependence of pH on the chemical shift.

To increase the sensitivity in 13C experiments, we developed a hyperpolarization protocol for ZA. Results in vivo in kidneys and tumors (Figure 2) clearly demonstrate that ZA represents a feasible novel candidate for pH imaging with advantages over existing methods. The demonstrated robustness of using ZA for extracellular pH imaging, its lack of toxicity and its accuracy render this new pH imaging technique promising for further development.

Figure 2: Hyperpolarized ZA in vivo pH measurements show an acidic tumor pH at 7 T. Representative tumor data from a hyperpolarized 13C measurement (colored) in an axial slice overlayed on anatomical proton images (grayscale). A calibration phantom containing 13C urea and the catheter used for injection are visible. Both (a) ZA and (b) urea accumulate in the MAT B III tumor. (c) The mean pH map shows a lower pH value in the tumor compared to the surrounding tissue. For all 13C images, intensity windows are based on sufficiently high signal levels for either (intensity images) or both (pH images) ZA and urea. Spectra from tumor voxels (d), shown for one representative animal) are best fitted with two pairs of ZA peaks (red, blue) and consistently show different pH values in the tumor in comparison to the vena cava, demonstrated in five animals (e) individual datapoints and mean ± s.d.) and compared with three other interstitial/extracellular pH measurements in four animals, namely 31P MRS in vivo using 3-APP, a needle-type optical sensor in vivo and an ex vivo tissue electrode pH measurement. Scale bars, 1 cm. In another recent study, we show that hyperpolarized and deuterated [1,5-13C2, 3,6,6,6-D4]zymonic acid (ZAd) can be used as an even more sensitive in vivo biosensor to non-invasively image pH. Furthermore, we demonstrate real-time imaging of dynamic pH changes in vitro.